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  • Bruno Vellutini 16:37 on 2015/08/25 Permalink
    Tags: confocal,   

    Membranipora simple staining 

    Just a simple staining of mixed stages of Membranipora using DAPI and Phalloidin 647 for morphology.

    • 3x 30min 0.2% PTx washes.
    • 1x PTx overnight at 4°C.

    26/08/15

    • Staining with Phalloidin and DAPI for 2h.
    • Let embryos in PBS+DAPI 1:1000 at 4°C.

    It failed… weird.

     
  • Bruno Vellutini 11:47 on 2015/08/18 Permalink
    Tags: confocal, , , gata, , , nk2.1,   

    Membranipora MAPK staining on in situ embryos 

    Selected 5 genes for a MAPK immuno staining to resolve the spatial relationship between MAPK vegetal blastomere, gene expression and the embryonic axes.

    gata_b foxa
    six3/6 evx
    nk2.1

    I started to de-glycerol embryos in hourly washes of PTw around 1200.

    20/08/2015

    • 3x 10min PTx
    • 2x 45 PBT
    • 2x 30min PTx+NGS
    • 1:200 anti-mapk antibody added at 14:00

    21/08/2015

    • Wash primary antibody (afternoon)
    • 3x 5min.
    • 4x 30min.
    • Add secondary antibody anti-mouse pod at 1:250 dilution at 1800.

    22/08/2015

    • 3x 5min PBT washes.
    • 4x 30min PBT washes.
    • Overnight at 4°C in PBT.

    23/08/2015

    • Washed 3x 5min in TNT buffer.
    • Developed in 50 µL TSA + Cy5 fluorochrome for 3 min.
    • Washed 10min in detergent solution at 67°C and another with warm detergent but at room temp to cool down.
    • Washed 2x 5min PBS.
    • TDE series ending with 97% TDE in PBS with DAPI 1:1000.

    24/08/2015

    Mounted slides and checked under scope. There is quite some background with the orange filter for cy3. I cannot distinguish any signal at the posterior vegetal blastomere. Confocal showed that there was no signal from the MAPK antibody. Maybe the longer time in glycerol messed the epitopes? Or the time developing (3min) was not enough…

    DAPI (cyan), MAPK (magenta), in situ (yellow):

     
  • Bruno Vellutini 18:01 on 2015/02/26 Permalink
    Tags: confocal, , ,   

    Immuno staining for azak and dmh1 treated Terebratalia 

    Primary: Tyros. Tub. 1:500 (a594 mouse) / Synapsin II 1:500 (a647 rabbit).

    Secondary: Alexa 594 (mouse) / Alexa 647 (rabbit) in a concentration of 1:200.

    Staining: DAPI 1:500/ PHALL for 2h.

    Mid blastula

    1µM 10µM
    control  dmh1 treated

    Early gastrula

    1µM 10µM
    control  dhm1 control

    Mid blastula to larva

    1µM 10µM
    control
    • Washed and cleaned embryos many times ~1h.
    • 1600 BSA 2x
    • 1730 NGS
    • 1830 Incubate

    27/02/2015

    • 3x 5 min 0.2% PTx+BSA
    • 4x 30 min 0.2% PTx+BSA
    • 2h 0.2% PTx+NGS
    • Added A594 (mouse) and A647 (rabbit) 1:200.

    28/02/2015

    • 3x 5 min 0.2% PTx+BSA
    • 4x 30 min 0.2% PTx+BSA
    • 2h staining in BODIPY FL and DAPI
    • 4x 5min 1x PBS

    01/03/2015

    Mounted in Murray and confocal-ed.

     
  • Bruno Vellutini 18:52 on 2015/02/13 Permalink
    Tags: confocal, , , serotonin, , synapsin, , tyrosinated tubulin   

    Brachiopod mesoderm immunostaining 

    Mesoderm and maybe some neurons and muscles. Engrailed antibody second try
    Ttra mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Ttra mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    Nano mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Nano mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    • 2h permeabilizing in 0.2 % PTx
    • 2h 0.2 % PTx + BSA.
    • 1h 0.2 % PTx + 5 % NGS.
    • Incubated with primary antibodies at 4°C in 0.2% PTx+NGS.

    14/02/15

    • Washed PTx+BSA 3x 5 min.
    • Washed PTx+BSA 4x 30 min.
    • Incubated 1h in PTx+NGS.
    • Added secondaries (alexa 594 mouse and alexa 647 rabbit) in a concentration of 1:200.

    15/02/15

    • Washed PTx+BSA 3x 5min.
    • Washed PBT 5x 10 min.
    • Stained with BODIPY FL and DAPI (1:500) for 2h.
    • Washed out staining with 1x PBS.
    • Overnight at 4 °C.

    16/02/15

    Mounting with Murray Clear went well.

     
  • Bruno Vellutini 21:02 on 2014/07/15 Permalink
    Tags: confocal, ,   

    Anti-MAPK immuno staining Membranipora 

    I realized that I don’t have a proper description of all Membranipora stages with the anti-mapk antibody. I prepared 4 wells with mixed stages as following:

    S1: 3h, 10h, 11h, 13h, 16h S2: 18h, 22h, 26h, 30h, 42h
    S3: 46h, 50h, 68h, 70h, 88h S4: 3d, 4d, 5d, 9d
    1. Mix samples in PTx.
    2. Exchange 4x in 30 min.
    3. 2x 15 min PBT.
    4. 1x 40 min PTx+NGS.
    5. Incubate with Anti-MAPK 1:200 in 200 µL of PTx+NGS.

    16/07/14

    1. 3x 5 min PBT.
    2. 4x 30 min PBT.
    3. 30 min PTx+NGS.
    4. 1:250 Anti-Mouse POD (Jackson) + Anti-Mouse HRP in PTx+NGS and incubate overnight in cold room at 21h.

    17/07/14

    1. Washed out secondary with 3x 5min and 4x 30 min PBT.
    2. Washed 3x 10min in TNT buffer.
    3. Develop with TSA+Cy5 for 5 min.
    4. Stopped the reaction in PTx.
    5. 45 min with 1:4000 Sytox Green.
    6. Embedding with TDE.

    I realized that sytox staining was fading very quickly under fluorescent scope and decided to add 1:2000 sytox green into TDE and leave it overnight.

    18/07/14

    Mounted and scanned sample 1 only in the confocal. They look nice, but maybe next time I would either stop the reaction with detergent solution or develop for shorter time, 3 minutes maybe.

    MAX_Mmem_MAPK_1.lif - Series003 MAX_Mmem_MAPK_1.lif - Series016 MAX_Mmem_MAPK_1.lif - Series022

     
  • Bruno Vellutini 18:52 on 2014/06/14 Permalink
    Tags: confocal,   

    Live membrane staining in Membranipora 

    I decided to try the live membrane staining FM® 4-64FX, fixable analog of FM® 4-64 membrane stain with Membranipora embryos on the confocal. I dissolved the dessecated FM into 100 µL of DMSO and then diluted 2.5 µL in 500 µL of FSW (final concentration of 5µg/mL). I then added embryos to a slide and completed the volume with the staining solution and sealed as for the 4D. Using the 561 laser I get a really nice signal and I did a few xyzt stacks to test. They look nice!

     
  • Bruno Vellutini 00:12 on 2014/05/13 Permalink
    Tags: confocal,   

    Confocal with Membranipora propidium iodide and phalloidin far red 

    Round of confocal with wild type Membranipora embryos. I mistakenly stained with propidium iodide instead of sytox green, but well. They were mounted in SlowFade to see if this is better than glycerol. However, it seems that it is incompatible with propidium and the nuclear staining became a general cytoplasm staining.. Many interesting scans of early and late gastrulation.

    MAX_Mmem pi-phallred.lif - Series012

     
  • Bruno Vellutini 19:30 on 2014/04/10 Permalink
    Tags: confocal, ,   

    Membranipora staining with Phalloidin 647 and Sytox Green in TDE 

    I scanned the U0126 treated embryos with DAPI+Phallacidin in glycerol and the outcome was far from optimal. Penetrance was not great and DAPI is always week… So I decided to try to get a nice morphological staining using TDE, to scan the whole sample without loss; Sytox Green, for strong nuclear staining; and Phalloidin 647, for cell linings and actin. I think this should give a clear picture and nice composition for a morphological stance.

    I selected different stages from cleavage to cyphonautes and added to 2 wells. In one I’m doing the staining above and in the other I’m just mounting them in TDE without phalloidin or sytox. I want to clarify if they have auto-fluorescence at all when embedded in TDE. I suspect they have it on the green.

    Phalloidin + Sytox Nothing, just unstained embryos

    I got 6.25 µL of Phalloidin 647, dried in a tube and diluted in 250 µL of 0.1% PTx with 1:1000 dilution of Sytox Green. Incubated covered for 1h40 and washed 4x 5 min in PBS. Kept the well in the dark at 4 °C.

    11/04/14

    Embed samples in TDE and mount slides to check signal.

    12/04/14

    Confocal?

     
  • Bruno Vellutini 10:36 on 2013/12/16 Permalink
    Tags: confocal, , ,   

    Propidium+Phallacidin and Murray Clear for Novocrania engrailed fluorescent in situ 

    Since mounting with TDE was not so good (crystals and some weird noisy background) I am staining with propidium iodide and phallacidin and mounting with murray clear. This should give a clean embryo with the mesodermal signal visible enough for details. Fluorochrome for engrailed is Cy5.

    17/12/13

    Confocal.

     
  • Bruno Vellutini 10:16 on 2013/11/18 Permalink
    Tags: confocal, ,   

    Phallacidin staining for Novocrania and Membranipora 

    Stain Novocrania and Membranipora with phallacidin then mount in TDE and glycerol, respectively (phallacidin has to be working in Membranipora to help with the phenotype description).

    Novocrania to be mounted in TDE:

    Novocrania mixed stages

    Membranipora to be mounted in glycerol:

    Mmem control Mmem U0126 1µM
    Mmem U0126 10µM Mmem U0126 25µM

    Put in TDE and glycerol today.

    19/11/13

    Mount slides for confocal:

    • Novocrania mixed stages in TDE+DAPI.
    • Membranipora each treatment in glycerol+DAPI.
    • Novocrania failed in situs in glycerol+DAPI for morphology.

    20/11/13

    Confocal day.

     
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